Lipophilic diesters of chelating agent for inhibition of enzyme activity

ABSTRACT

The present invention relates to the use of lipophilic diesters of the chelating agent 1,2-bis(2 aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) for inhibition of proteolytic activities of certain metalloproteinases and of calpain. The invention further relates to methods for preventing, treating or managing MMP-related and calpain-related diseases or disorders in mammals comprising administering to a mammal in need thereof, a pharmaceutical composition comprising a therapeutically effective amount of said lipophilic diesters of the chelating agent BAPTA.

FIELD OF THE INVENTION

The present invention relates to the use of lipophilic diesters of thechelating agent 1,2-bis(2 aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA) for inhibition of proteolytic activities of certainmetalloproteinases and of calpain.

BACKGROUND OF THE INVENTION

Matrix metalloproteinases (MMPs) are extracellular zinc- andcalcium-dependent proteases, which are produced in a latent form andrequire activation for catalytic activity. Activation occurs at the cellsurface and enables MMPs to degrade components of the extracellularmatrix (ECM) at specific sites in the membrane surroundings. The moststudied MMPs are the gelatinases, which include MMP-2 and MMP-9 that usegelatin, Type IV collagen and fibronectin as preferred substrates. Whiletranscription of MMP-9 genes is transactivated by cytokines and growthfactors, MMP-2 is constitutively expressed and unresponsive to phorbolester and most cytokines. MMP-2 activation is regulated by MT1-MMP,which is a membrane-anchored MMP. MMP-9 activation is regulated by aprotease cascade involving plasmin and stromelysin-1 (MMP-3).

Matrix metalloproteinases are responsible for much of the turnover ofmatrix components and as such are involved in normal as well as inpathological processes. MMPs have an important role in maintenance andremodeling of membranes and ECM, for example, in breaking down theextracellular matrix to allow cell growth and tissue remodeling duringdevelopment and recovery from injury. They also play a role in processessuch as ovulation, modulation of capillary permeability and in enablingcell migration to a site of inflammation.

MMPs are involved in many pathological conditions. For example, they areassociated with pathogenic mechanisms in cancer such as invasion,metastasis and angiogenesis [Reviewed by Foda and Zucker (2001) DrugDiscovery Today 6: 478-482]. MMPs play a part in progression ofinflammatory conditions and diseases involving degradation ofextracellular matrix, such as in stroke, hemorrhage, rheumatic diseases(e.g. arthritis), Crohn's disease, asthma, and in cerebrovascular andcardiovascular disorders [Mun-Bryce and Rosenberg (1998) J. CerebralBlood Flow Metabolism 18:1163-72; Yong et al. (1998) TINS 21:75-80;Lukes et al. (1999) Mol. Neurobiol. 19:267-284]. Members of the MMPfamily have also been implicated in neurological diseases and conditionsas being involved in demyelination and neuro-inflammatory processes. Forexample, MMPs have been associated with brain damage and ischemia,Guillain-Barré, multiple sclerosis, amyotrophic lateral sclerosis andAlzheimer's disease. The current notion is that inflammation leads tothe production of cytokines, chemokines, growth factors and hormonesthat modulate MMPs production. Activation of MMPs and plasminogenactivators (PAs) is an important regulatory step in the inflammatoryresponse.

Members of another family of metalloproteinases, identified as “ADisintegrin And Metalloproteases” (ADAM), have, like members of the MMPfamily, multiple domains including a zinc-dependent catalytic domain anda N-terminus pro-domain that is responsible for maintaining the enzymein an inactive state [Moss et al. (2001) Drug Discovery Today 6:417-426]. It was shown that members of the ADAM family are involved inseveral different processing events including cleavage of substrates offthe cell membrane surface (a phenomenon termed “shedding”). One memberof the ADAM family is the TNFα-Converting Enzyme (TACE). TACE is foundon the cell surface where it processes the membrane-bound TNFα, apro-inflammatory cytokine, to its mature soluble form. Soluble TNFα,which is released in inflammatory conditions, can induce apoptosis. Forexample, TNF induces secretion and activation of MMP-9 in macrophagesand glial cells and causes neuronal cell death in neuroinflammatorydiseases and following brain injuries. TNF has also been shown to play arole in pathological conditions such as rheumatoid arthritis.

As potentially highly toxic proteolytic enzymes, the matrixmetalloproteinases are tightly regulated at multiple stages, as follows:

i) Gene transcription—most MMPs are not constitutively expressed, buttheir transcription is controlled by various cytokines (e.g. IL-1, TNF)and growth factors (e.g. TGF-β, retinoic acid, FGF).

ii) Pro-enzyme activation—MMPs are normally produced in a latent form(pro-MMP) including a propeptide segment that generally must be removedto activate the enzyme.

iii) Inhibition of enzyme activity—There are at least four endogenousMMP-inhibitors known as tissue inhibitors of metalloproteinases (TIMPs),which bind to the enzyme and block its activity. Another known naturalinhibitor of MMPs is the serum proteinase inhibitor α-macroglobulin.

Several synthetic inhibitors of MMPs have been described in variouspublications in the scientific and patent literature. Currently knowninhibitors mainly include synthetic peptides and chelating agents[reviewed by Woessner J F Jr. in Ann N.Y. Acad. Sci. (1999) 30:388-403].

Some synthetic inhibitors of the MMP active site are peptidomimeticsbased on the sequence of peptides cleaved in collagen [Masui et al.(1977) Biochem Med. 17:215-21). Peptidic agents based on conservedpeptide sequence derived from the pro-segment of human collagenase IVare disclosed by Stelter-Stevenson et al. [Am J Med Sci. (1991)302:163-70] and in U.S. Pat. No. 5,270,447 to Liotta et al. Syntheticpeptides isolated from phage display peptide libraries and cyclicpeptides with MMP inhibitory activity are described by Koivunen et al.[Nat. Biothechnol (1999) 17: 768-74].

N-hydroxyformamide peptidomimetics useful as TACE and MMPs inhibitorsare disclosed by Musso et al. [Bioorg Med Chem Lett (2001) 11: 2147-51].

Other polypeptides and peptoid compounds useful as metalloproteinaseinhibitors are disclosed in U.S. Pat. Nos. 4,263,293 and 4,297,275 toSundeen et al., in U.S. Pat. Nos. 4,371,465, 4,371,466 and 4,374,765 allissued to McGregor, and in U.S. Patent Publication No. 2002/0090654 toLangley et al.

Non-peptidic MMP-inhibitory compounds are disclosed in U.S. Pat. No.4,950,755 to Witiak et al. and in U.S. Pat. No. 5,866,570 to Liang etal.

MMPs inhibitors comprising targeting moieties and chelators aredisclosed in International Patent Publication No. WO 01/60820 of DupontPharmaceuticals Company, and in International Patent Publication No. WO02/053173 to Kimberly-Clark Worldwide, Inc.

Matrix metalloproteinases as well as other members of the ADAM familyare inhibited by chelating agents. Most of these chelating agents arenatural and synthetic hydroxamate compounds and derivatives thereof suchas succinyl hydroxamate, sulfonamide hydroxamate etc. [reviewed byWoessner, J. F. Jr. (1999) in Annals New York Academy of Sciences 30:388-403]. For example, the synthetic hydroxamates batimastat (BB-94;Invest New Drugs (1996) 14:193-202) and its orally bioavailable analoguemarimastat have been shown to inhibit spread and growth of malignanttumors in animals. These compounds are currently examined in advancedclinical trials.

Among the compounds that have been shown as MMPs inhibitors are alsoantibiotics such as tetracyclines and their chemically modified analogs(Golub et al. (1983) J Periodontal Res. 18:516-26; U.S. Pat. No.4,704,383 to McNamara et al.; U.S. Pat. No. 5,837,696 to Golub et al.].

Most of the above-mentioned agents are non-specific inhibitors ofmetalloproteinases and other metal-ion dependent proteases.

Calpains are members of another family of proteases. These are cytosolicenzymes which are calcium-dependent cysteine proteases. Calpainspredominantly exist within cells as inactive proenzymes and areconverted into their active forms in the presence of elevatedintracellular calcium levels. Upon binding of calcium, the precursorenzyme goes through a self-digestion process that results in release ofthe activated calpain.

A wide range of proteins serves as substrates for calpain includingcytoskeletal, membrane and regulatory proteins. Calpain participates ina number of normal cellular signal transduction systems as well as inpathological conditions. For example, calpain activation has beenassociated with ischemia and neuronal cell death such as those caused bystroke and traumatic brain and spinal cord injuries [Bartus et al.(1995) Neurol. Research 17:249-258]. Calpain proteolytic activity hasalso been implicated in several neurodegeneration diseases andconditions, including Alzheimer's Disease, Parkinson's disease,Huntington's disease and Pick's disease.

Presently known natural and synthetic calpain inhibitors, including bothpeptidic and non-peptide molecules, are reviewed by Wang and Yuen[“Calpain inhibition: an overview of its therapeutic potential” inTrends Pharm. Sci. (1994) 15, 412-419] and by Donkor [“A survey ofcalpain inhibitors” in Curr. Med. Chem. (2000) 7:1171-88]. Known calpaininhibitors include polypeptides which mimic peptide sequences of thenatural inhibitors calpastatin and kininogen [for review, see Wang andYuen (1994) Trends Pharm. Sci. (1994) 15, 412-419].

Compounds which are sulfonamide derivatives and ketone derivatives thatpossess inhibitory activity against cysteine proteases are disclosed,respectively, in U.S. Pat. Nos. 5,506,243 and 5,639,783 both to Ando etal. Calpain inhibitors which are di-peptide alpha-keto esters,alpha-keto amides and alpha-keto acids are described by Li et al. [J.Med Chem (1993) 36: 3472-80]. Several other classes of calpaininhibitors are disclosed by Bartus et al. in International PatentPublication no. WO 92/11850.

Most commercially available calpain-inhibitors are compounds based onpeptide structures that interact with the substrate-binding site of theenzyme. Many of these compounds are non-specific and inhibit a widevariety of proteases in addition to calpain. Moreover, most of the knowninhibitors that were active in vitro, were found ineffective ininhibiting calpain in-vivo, in particular in the CNS, as being poormembrane permeants. Furthermore, almost all MMPs-inhibitors tested fortreating pathological inflammatory conditions or cancers failed inin-vivo clinical studies.

Thus, there remains a long-felt need for effective, non-toxic agentswhich are specific inhibitors of critical proteases such as the MMPs andcalpain.

BAPTA-Diesters

Stable lipophilic diesters of the divalent metal ion chelator 1,2-bis(2aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) have beendisclosed in the International Patent Publication No. WO 99/16741 of thesame applicant. Also disclosed in this publication is the use of thesecompounds in pharmaceutical compositions useful for treating diseasesand disorders related to excess of divalent metal ions. Among thesediseases and disorders are ischemia, stroke, epilepsy andneurodegenerative diseases such as Alzheimer's disease and Parkinson'sdisease.

At that time, however, the mechanism by which these chelating agentsexert their neuroprotective effects has not been elucidated ordisclosed. No indication or suggestion for the cellular targets affectedby these chelators has been mentioned in the WO 99/16741 or any otherpublication.

SUMMARY OF THE INVENTION

It has now been found, in accordance with the present invention, thatcertain diesters of the chelating agent 1,2-bis(2aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (hereinafter denoted as“DP-BAPTAs”) are capable of inhibiting enzymatic activities of theproteases matrix metalloproteinase (MMP), calpain and TNFα-ConvertingEnzyme (TACE).

Accordingly, the present invention provides, in one aspect, a method ofinhibiting protease activity, said protease being selected frommetalloproteinase and calpain, the method comprising exposing cells toinhibiting amount of a compound of the general formula (I):

whereinR is saturated or unsaturated alkyl, cycloalkyl, arylalkyl orcycloalkyl-alkyl radical having from 1 to 28 carbon atoms which may beinterrupted by any combination of 1-6 oxygen and/or nitrogen atoms,provided that no two oxygen atoms or an oxygen and a nitrogen atom aredirectly connected to each other; and M denotes a hydrogen or aphysiologically acceptable cation.

In addition, certain compounds disclosed herein are novel and inthemselves constitute an aspect of the present invention. Thesecompounds include the compound of the general formula (I) wherein R is2-benzyloxyethyl. Thus, according to another aspect of the invention,there is provided a compound of the general formula (I) which is1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-benzyloxyethyl acetate),N,N′-acetic acid and salts thereof. Also provided by the invention arepharmaceutical compositions comprising a therapeutically effectiveamount of said compound and a pharmaceutically acceptable carrier orexcipient.

According to currently preferred embodiments of the invention, theuseful compounds for inhibiting the MMP-9 activity are the followingcompounds and physiologically acceptable salts thereof:

1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-octoxyethyl acetate),N,N′-diacetic acid (denoted herein DP-b99),

1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-octodecyloxyethyl acetate),N,N′-diacetic acid (denoted herein DP-b109),

1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-benzyloxyethyl acetate),N,N′-acetic acid (denoted herein DP-b440),

1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-dodecyloxyethyl acetate),N,N′-diacetic acid (denoted herein DP-b460),

1,2-bis(2-aminophenoxy)ethane, N,N′-di[2-(2-dodecyloxyethoxy)-ethylacetate], N,N′-diacetic acid (denoted herein DP-b458), and

Currently most preferred compounds for inhibiting calpain activity are1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-octoxyethyl acetate),N,N′-diacetic acid (DP-b99) and 1,2-bis(2-aminophenoxy)ethane,N,N′-di(2-octodecyloxyethyl acetate), N,N′-diacetic acid (DP-109) andphysiologically acceptable salts thereof.

In another aspect of the invention, there are provided methods forpreventing, treating or managing diseases and pathological conditionsassociated with damaging MMP and/or calpain activities. The methodscomprise administering to a mammal in need thereof, a pharmaceuticalcomposition containing as an active ingredient a therapeuticallyeffective amount of a compound of the above-mentioned general formula(I).

In yet another aspect of the invention, there is provided the use of acompound of the general formula (I) for the preparation of a medicamentfor inhibiting the activity of a protease selected frommetalloproteinase, calpain and TACE.

The present invention further provides methods and the use of thecompounds of the general formula (I) for the treatment of MMP- orcalpain-related diseases, disorders or conditions, which may be selectedfrom the group consisting of cancer (including metastasis cancer),angiogenesis-dependent diseases (e.g. cancerous tumors, arthritis,psoriasis, macular degeneration, chronic inflammation and diabeticretinopathy), ischemic or hypoxic tissue damage, oxidative injury,stroke, trauma, inflammatory conditions and diseases (e.g. arthritides,rheumatoid arthritis, osteoarthritis, restenosis, asthma, psoriasis,systemic lupus erythematosus, inflammatory bowel syndrome, Crohn'sdisease, gingivitis, periodontitis, meningitis, tropical spasticparaparesis, sepsis, bullous skin disorders, acne and inflammation dueto infectious diseases), atherosclerosis, thrombotic disorders,arthritis, osteoporosis, diabetes, hemorrhage, autoimmune diseases,rheumatic diseases, ocular pathologies and retinopathies (e.g. diabeticretinopathy, glaucoma, macular degeneration, cataract, retinaldetachment and retinal tears), burns, chronic wounds (e.g. ulcers),neurological and neurodegenerative diseases and disorders (e.g. multiplesclerosis (MS), Alzheimer's disease (AD), motor neuron disease (MND),amyotrophic lateral sclerosis (ALS), Guillain-Barré, Parkinson'sdisease, Huntington disease, Pick's disease, dementia syndrome, vasculardementia, multiple infarct dementia, HIV-induced neural disorders, brainischemia (both global and focal ischemia) and neuronal tissue trauma),migraine, cerebrovascular and cardiovascular disorders.

The methods of treatment in accordance with the invention may furthercomprise treating the patient with additional therapeutic treatmentwhich may be carried out concurrently with, preceding or subsequent tothe administration of the pharmaceutical composition comprising acompound of the general Formula (I).

It is important to note that not any chelator can inhibit the activityof the tested proteases. In contrast to the effect of DP-BAPTAcompounds, the closely related known chelators, BAPTA and BAPTA-AM, atdoses similar to DP-BAPTA, did not inhibit MMP-9 activity. In fact,BAPTA-AM even slightly enhanced the MMP-9 activity.

Another point to emphasize is that, under the experimental conditionsemployed, the tested DP-BAPTAs inhibited calpain and MMP-9 proteolyticactivities, however no such effect could be demonstrated for the othergelatinase tested, MMP-2.

Further objects of the present invention will become apparent to thoseskilled in the art upon further review of the following disclosure,including the detailed descriptions of specific embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depict total amount of MMP-9 enzyme (A) and the MMP-9enzymatic activity (B) in the conditioned media collected from culturedA-172-glioma cells treated with 10 ng/ml TNFα, in the absence orpresence of 20 μM or 50 μM DP-b109, as indicated.

FIGS. 2A-B depict gelatin-zymogram gels used for detection of MMP-9activity in conditioned media collected from cultured C6-glioma cellsthat were treated with 20 ng/ml TNFα (A) or 0.1 μM PMA (B), either inthe absence or presence of different concentrations of DP-b109 asindicated. Areas of MMP-9 and MMP-2 protease activity, showed as clearbands, are marked with arrows.

FIGS. 3A-C depict a representative gelatin-zymogram gel (3A) andanalysis of biologically active MMP-9 (3B) and pro-MMP-9 (3C) forms ofthe enzyme. Conditioned media collected from C6-glioma cell culturestreated with 10 or 20 ng/ml TNFα, either in the absence (white bars) orpresence (dark bars) of 25 μM DP-b99, as indicated, were run on zymogramgels. Arrows mark the pro- and active MMP-9 bands on the gel (3A). Bandanalyses (3B and 3C) were performed using the Kodak digital Science™ 1DImage Analysis software.

FIG. 4 depicts TNFα release from primary glial cells treated with 0.5μg/ml LPS in the absence or presence of different DP-BAPTA compounds asindicated.

FIGS. 5A-B depict a gelatin-zymogram gel (A) and MMP-9 band analysis (B)of lysates of the right (R-injured) or left (L-intact) hemispheres frombrains of unilateral MCAO treated rats. The animals were treated, 2hours after reperfusion, with either 5 μg/kg DP-b99 or vehicle only, asindicated.

FIG. 6 depicts a Western blot of cortical primary cells lysates reactedwith anti-spectrin antibodies as described in Example 11. The cells werepre-treated with either DP-b99 (15 μg/ml) or the commercial calpaininhibitor MDL28170 (25 μM) for one hour prior to induction of oxidativestress (H₂O₂, 50 μM) as indicated. The non-cleaved (280 kDa) andcalpain-cleaved (150 kDa) forms of spectrin are marked by arrows.

DETAILED DESCRIPTION OF THE INVENTION

The synthesis and some utilization of stable lipophilic diesters ofBAPTA (DP-BAPTAs) have been disclosed in the International PatentPublication No. WO 99/16741 of the same applicant, the teaching anddisclosure of which are expressly incorporated herein in their entiretyby reference. In the WO 99/16741 publication, the neuroprotectiveeffects of DP-BAPTAs were demonstrated in neuronal cell culturesin-vitro, and in ischemia model systems in-vivo. However, the effect ofthe DP-BAPTA molecules on activities of specific enzymes has not beentaught or suggested in that or any other publication. Accordingly, itwas neither taught, recognized or suspected that these compounds couldbe effectively use for the treatment of MMP- and calpain-relateddiseases and disorders as disclosed in the present application.

It is now disclosed, for the first time, that certain diesters of thechelating agent BAPTA are capable of inhibiting the activity of calpainand of certain proteases of the ADAM family, and in particularinhibiting the activity of matrix metalloproteinase-9 (MMP-9).

The useful compounds in accordance with the invention are of the generalformula (I) as described above. It is to be understood that within thescope of the invention are included also pharmaceutically acceptablesalts of the compounds of the general formula (I) including organic andinorganic cations, as well as various solvates, including hydrates, andother active forms of the compounds of the general formula (I).

Currently preferred compounds for inhibiting MMP or calpain activitiesare diesters of BAPTA with alkyl chains comprising from around 8 to 20carbon atoms. The alkyl chains may be saturated or unsaturated alkylsincluding one or more double bonds and/or a triple bond. According topreferred embodiments of the invention, the alkyl chain is interruptedby from 1 to 3 oxygen atoms. According to most preferred embodiments,the R moiety of a compound of the general formula (I) includes amonoalkyl ether of ethylene glycols, preferably mono-, di- ortri-ethylene glycols.

The alkyl residue at position R of the compound of the general formula(I) may consist of or include cyclic elements that may be aromatic ornon-aromatic ring structures. Preferably the cyclic elements have 5 or 6carbon atoms.

Currently preferred cyclic R radicals comprise aromatic ring which is aphenyl residue. Other currently preferred cyclic elements included atposition R are saturated or unsaturated cyclopentyl, cyclohexyl orcycloheptyl. The cyclic elements may be directly linked to the carboxymoiety of the compound of the general formula (I), or linked via asaturated or unsaturated alkyl chain that may include one or more oxygenand/or nitrogen atoms.

Preferably, the compound of the general formula (I) includes amonovalent cation at position M. Suitable pharmaceutically acceptablecations may include, but are not limited to, H⁺, Na⁺, Li⁺, K⁺, NH₄ ⁺ andmono-alkylammonium. Also divalent cations may be included at position M.The choice of the preferred cation at position M of the general formula(I) depends on the intended therapeutic use of the compound, as well ason the specific formulation and route of administration employed. Aperson skilled in the art will be able to select the appropriate cationas required for the optimal pharmaceutical compositions and way ofadministration chosen in each particular treated case.

One of the most preferred DP-BAPTA compounds is1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-benzyloxyethyl acetate),N,N′-acetic acid (DP-b440), where the R moieties of the compound of thegeneral formula (I) include an alkylaryl moiety. This compound wasdisclosed generally in the WO 99/16741 publication, but was notspecifically claimed and not individually tested.

It has now been shown by the inventors of the present invention thatDP-BAPTAs can attenuate or block both basal MMP-9 activity and TNFα- orPMA-induced activation of MMP-9. DP-BAPTAs can also inhibit calpainactivity. Hence, DP-BAPTAs may be useful in reducing deleteriousprotease activities in pathological conditions due, for example, toischemia and inflammatory responses. Accordingly, DP-BAPTA compounds maybe useful in preventing, treating or managing diseases and pathologicalconditions associated with harmful activities of matrixmetalloproteinases or calpains.

It is important to note that while MMP-9 activity was significantlyinhibited by the DP-BAPTA compounds, such inhibitory activity could notbe demonstrated with the related chelators tested, namely1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) or1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid acetoxymethylester (BAPTA-AM).

The DP-BAPTA compounds in accordance with the invention may be useful inthe treatment of a whole range of indications which involve degradationprocesses carried out or mediated by MMPs, TACE or calpain. Theseindications include, but are not limited to, diseases and conditions dueto neuronal ischemia (e.g. global brain ischemia and focal brainischemia), cardiac ischemia, trauma, stroke and inflammatory conditionsincluding neuroinflammatory diseases and disorders, rheumatic andautoimmune diseases, neurological, cerebrovascular and cardiovasculardiseases and disorders. The DP-BAPTA compounds may also be useful incompositions and methods for enhancing wound healing such as, forexample, in burns and in chronic wounds (e.g. ulcers).

A large amount of data has been accumulated which show MMP- and/orcalpain-involvement in progression of diseases and conditions whereinflammatory processes are implicated. These pathological conditionsinclude, but are not limited to, rheumatoid diseases (e.g. rheumatoidarthritis and osteoarthritis), asthma, psoriasis, systemic lupuserythematosus, inflammatory bowel syndrome, Crohn's disease, gingivitis,periodontitis, meningitis, tropical spastic paraparesis, sepsis, bullousskin disorders, acne and inflammation due to infectious diseases.

The infectious diseases may include, but are not limited to, infectiousdiseases caused by any type of microorganism such as bacteria, fungi(e.g. candidiatis, aspergilosis) and viruses (e.g. herpesviruses-related disorders, HIV-related diseases), by parasites (e.g.malaria, amebiasis) or by prions (e.g. Creutzfeld-Jacob Disease).

Inhibitors of MMPs or calpain can reduce proteolytic damage to tissuessuch as that caused during inflammatory processes. For example, maylimit brain-blood-barrier (BBB) breakdown, inhibit neuroinflammation(e.g. as in meningitis), reduce damage associated with brain or cardiacischemic injuries, and may diminish proteolytic effects caused byinsults such as oxidative stress, burns, infections and central (CNS)and peripheral nervous system (PNS) injuries due to physical causes(e.g. trauma).

Elevated MMP or calpain activity has been linked to severalneurodegenerative diseases and conditions including, but not limited to,multiple sclerosis (MS), motoneuron disease (MND), amyotrophic lateralsclerosis (ALS), Alzheimer's disease, Guillain-Barré syndrome,Parkinson's disease, Huntington's disease, Pick's disease, dementiasyndrome, vascular dementia, multiple infarct dementia, HIV-inducedneural disorders, brain ischemia (both global and focal ischemia) andneuronal tissue trauma.

MMPs have also been associated with pathological conditions such asischemic or hypoxic tissue damage, oxidative damage, osteoporosis,hemorrhage, arterial restenosis, cardiovascular disorders (e.g. ischemicmyocardiac infarction) as well as with various ocular pathologies andretinopathies including diabetic retinopathy, glaucoma, maculardegeneration, cataract, retinal detachment and retinal tears.

Cancer is another major disease where it has been shown that proteolyticactivities of metalloproteinases contribute to the progression of thedisease. MMPs are involved in spread of cancer, and in particularfacilitating the metastasis state of the disease. MMP-2 and MMP-9 areinvolved in the breakdown of Type IV collagen, which is a majorcomponent of basement membrane, and as such may be key factors inprocesses involving membrane degradation, for example, in angiogenesisand in tumor invasion and metastasis. Indeed, positive correlation hasbeen found between tumor progression and expression of members of theMMP family. For example, increased expression of MMP-2 and MMP-9 geneshas been associated with malignancies of gliomas.

A number of factors are important in the progression of malignancies.One of the crucial factors is angiogenesis, which is believed to befundamental for primary tumor growth, tumor progression and metastasis.The first step in the mechanism of angiogenesis involves degradation ofbasement membrane so to facilitate the growth of a new capillary sprout.Thus, degradation and remodeling of the ECM are essential processes forthe mechanism of angiogenesis, and methods of inhibiting these processesmay be beneficial in blocking angiogenesis and hence diminishingmalignancies.

Angiogenesis is also important in a number of other pathologicalprocesses, including arthritis, psoriasis, diabetic retinopathy, chronicinflammation, scleroderma, hemangioma, retrolental fibroplasia andabnormal capillary proliferation in hemophiliac joints, prolongedbleeding etc. MMP-inhibitors are expected to be useful for the treatmentof these angiogenic-associated diseases.

The inability to control metastasis presents a major problem, asmetastases are the leading cause of death in patients with cancer. Todate, there is no satisfactory treatment for preventing or limitingmetastasis growth. Thus, the use of the DP-BAPTA compounds in accordancewith the present invention for inhibiting MMPs, and in particular forinhibiting the MMP-9 protease activity, may be beneficial in thisrespect.

The cancer subjected to treatment with DP-BAPTAs may include any type oftumors and neoplastic growths that may be benign or malignant growthsincluding primary tumors as well as secondary tumors. The terms “cancer”and “neoplastic growth” are interchangeably used in the specificationand claims and mean to cover all kinds of pathological uncontrolled cellgrowths including invasive and non-invasive neoplasms, solid andnon-solid tumors, and including remote metastases.

The term “treatment” as used herein means to include therapeuticprocedures aiming at preventing, ameliorating, palliating, inhibiting ordelaying the onset and/or development and/or progression of apathological condition or improving its manifestations.

It should be understood that the therapeutic activity of the compoundsof the general formula (I), as disclosed and claimed herein, isirrespective of the exact mechanism of action of these compounds and isnot meant to be limited to any particular mode of action by which thesemolecules exert their beneficial effect(s).

In accordance with the methods of the invention, a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof the general Formula (I) is administered to a patient in need thereof.

The administered pharmaceutical composition may include a compound(s) ofthe general Formula (I) as the sole active ingredient, or may includesaid compound(s) in combination with one or more additional agents knownto be effective in the treatment of a particular disease or disorder.The compound(s) of the general Formula (I) and the additionaltherapeutically active agent(s) may be included in the samepharmaceutical composition or may be administered in separatecompositions. Furthermore, the use of DP-BAPTA in combination withanother therapeutically active agent (or another therapeutic means) maybe concurrently or not. The methods of the invention includeadministration of the DP-BAPTA compound(s) either at the same time,preceding or following exposure to the additional therapeutic agent orprocedure.

Additional agents that may be used in combination with the DP-BAPTAcompounds may be therapeutic and prophylactic drugs, hormones,immuno-modifying agents etc. and may include other chelating agents,proteins, peptides, carbohydrates, lipidic molecules, DNA and RNAsequences etc.

These agents may be selected from, but are not limited to,anti-neoplastic, anti-proliferative, anti-inflammatory, antibiotic,anti-viral, anti-microbial, anti-mycotic, anti-allergic, cardiovascularagents, anti-convulsant, anti-depressant, anti-psychotic, analgesic,neurological agents, neuroprotective agents and bioactive peptides andproteins such as neurotransmitters, immuno-modulators, growth factors,hormones, antibodies etc.

For example, in the case of treating cancer, the DP-BAPTA compound(s)may be used alone or in combination, concurrently or not, withadditional anti-cancer treatment. The additional anti-cancer treatmentmay include, but is not limited to, chemotherapy, irradiation therapy,immunotherapy, genetic therapy, surgery or any other anti-cancertreatment as known in the art. The additional treatment may be carriedout concurrently with or consecutively to the administration of thecompounds of the general formula (I), namely the additional treatmentmay be applied concurrently or successively, either preceding orsubsequent to the administration of the compound of the general formula(I). The time interval between the two treatments and the overallregimen will be determined by a person skilled in the art taking intoaccount the specific treated disease and the particular condition andresponse of the treated individual to the treatment.

Any anti-cancer drug that is suitable for use in chemotherapy proceduresmay be applied in combination with the compound of the general formula(I). Suitable anti-cancer drugs may include, but are not limited to,alkaloids (e.g. taxol, vinblastine, vindesine and vincristine),alkylating agents such as alkyl sulfonates, aziridines, ethylenimines,methylmelamines, nitrogen mustards (e.g. cyclophosphamide) andnitrosoureas, antibiotics and analogs (e.g. aclacinomycin, actinomycin,anthramycin, daunorubicin and doxorubicin), antimetabolites such asfolic acid analogs (e.g. Tomudex®), purine and pyrimidine analogs andplatinum complexes (e.g. carboplatin, cisplatin, miboplatin andoxaliplatin).

The combination treatment with additional therapeutic procedures may bebeneficial also in treatment of other diseases and disorders. Forexample, in treatment of inflammatory, neuro- or cardiovascularconditions where the administration of the DP-BAPTA compounds may be incombination with (either concurrently, preceding or subsequent to)surgery and/or treatment with another medicament or therapeutic agent(e.g. antibiotics, antibodies etc.) to remove or kill infectious agentsor other pathogenic elements.

It will be readily apparent to those of ordinary skill in the art that alarge number of other beneficial drugs, reagents, means or proceduresmay be useful in the treatment of particular pathological conditions.Pharmaceutical compositions including these therapeutically effectiveagents and methods applying them or other medical procedures are alsoincluded within the scope of the invention as compositions and methodsuseful in combination with the compounds of the general formula (I). Theexact protocol and the additional medicament or therapeutic procedureused, will be determined by a person skilled in the art taking intoconsideration the particulars of the specific medical condition treated,e.g. the stage of the disease or disorder, its severity and progression,as well as the condition of the patient.

The pharmaceutical compositions comprising the compound of the generalformula (I) may be in a liquid, aerosol or solid dosage form, and may beformulated into any suitable formulation including, but not limited to,solutions, suspensions, micelles, emulsions, microemulsions, aerosols,ointments, gels, suppositories, capsules, tablets, and the like, as willbe required for the appropriate route of administration.

Any suitable route of administration is encompassed by the inventionincluding, but not being limited to, oral, intravenous, intraperitoneal,intramuscular, subcutaneous, inhalation, intranasal, topical, rectal orother known routes. In preferred embodiments, the useful pharmaceuticalcompositions are orally or intravenously administered. The dose rangesare those large enough to produce the desired proteinase inhibitoryeffect. The dosing range varies with the specific DP-BAPTA used, thetreated pathological condition and the route of administration and isdependent on the additional treatment procedure, if such additionaltreatment is applied.

The dosage administered will also be dependent upon the age, sex,health, weight of the recipient, concurrent treatment, if any, frequencyof treatment and the nature of the effect desired. The specific dosage,regimen and means of administration will be determined by the attendingphysician or other person skilled in the art.

The Invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Synthesis of BAPTA Diesters of Alkyl or Alkylaryl andSalts Thereof

Synthesis of disodium or calcium salts of some diesters of1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (DP-BAPTA) wascarried out in three steps as follows:

Step 1. Preparation of an Anhydride of BAPTA:

Step 2. Preparation of BAPTA Diester:

Step 3. Preparation of Disodium or Calcium Salt of the Diester of BAPTA:

Step 1. Preparation of BAPTA Anhydride

BAPTA (24 gr., 0.05 mol), pyridine (8 gr., 0.1 mol) and acetic anhydride(95 ml, 1.0 mol) are introduced into a round-bottom single-neck flask(500 ml), equipped with a reverse condenser (water cooling) and magneticstirrer. The reaction mixture is heated at 90° C. for 5 hours withvigorous stirring by magnetic stirrer. The temperature is then decreasedto 50° C. and heating is continued at this temperature for 10 hourslonger. At the end of the 10-hour period the reaction mixture is cooledto room temperature and the precipitate is extracted by filtration. Theprecipitate is then washed four times with ethyl acetate (50 ml eachwash) and twice with ether (60 ml each wash). The precipitate is driedunder vacuum at 50° C. for 6-8 hours. The product is a BAPTA anhydride.Yield 80% (17.6 g.). White solid. M.p. 148-149° C.

Analyses: TLC. The compound decomposed in the course of analysis.

¹H NMR (C₆D₅NO₂), δ (ppm): 4.40 (s, 8H), 4.47 (s, 4H) and 6.85-7.01 (m,8H).

IR: 1762.9 cm⁻¹ (s), 1820.7 cm⁻¹ (s).

Elemental. C₂₂H₂₀O₈N₂. Calculated: C, 60.00%; H, 4.54%; N, 6.36%; Found:C, 59.60%; H, 4.66%; N, 6.20%.

Step 2. Preparation of Alkyl or Aryl Diester of BAPTA

The BAPTA anhydride of step 1 (10 g, 0.023 Mol) and correspondingabsolute alcohol (300 ml) are introduced, under argon atmosphere, into around-bottom single-neck flask, equipped with reverse condenser andmagnetic stirrer. The mixture is heated in an oil bath at 90° C. (formethyl and ethyl diesters at 70° C.) with vigorous stirring. After 6hours about half of the alcohol is distilled from the reaction mixture(high molecular alcohols are distilled under vacuum). The obtainedmixture is cooled to 0° C. and kept at this temperature for 5-8 hours.The precipitate is separated from the solution by filtration (glassfilter N4) under vacuum and is washed 3-4 times with about 40 ml ofethanol, followed by three washes (100 ml each) of ethyl acetate andfinally with three washes (150 ml each) of diethyl ether. The product isdried under vacuum for 8 hours.

The chemical/physical specifications of synthesized diesters of BAPTAare presented hereinbelow:

Ethyl diester of BAPTA. Yield 90% (11 g.). White powder. M.p. 161-162°C. TLC analysis. Silica gel 60 on aluminum sheet. Eluent is mixture ofchloroform with methanol and water (80:20:1.5 v/v). For indication thechromatogram is sprayed by the indicator spray and then is charred at350-400° C. Composition of indicator spray is 4-methoxybenzaldehyde (10ml), ethanol (200 ml), 98% H₂SO₄ (10 ml) and glacial acetic acid (2 ml).One spot. R_(f) 0.3.

¹H NMR (CD₃OD). δ (ppm): 1.05-1.11 (t, 6H), 3.91-4.00 (dd, 4H), 4.05 (s,4H), 4.14 (s,4H), 4.27 (s, 4H), 6.83-6.96 (m, 8H).

Elemental. C₂₆H₃₂O₁₀N₂. Calculated: C, 58.64%; H, 6.03%; N, 5.26%.Found: 58.00%; H, 6.00%; N, 5.09%.

Propyl diester of BAPTA. Yield 90% (11.5 g.). White powder. M.p. 187° C.TLC analysis. Conditions of the analyses of diethyl and dipropyl estersof BAPTA are analogous. One spot. R_(f) 0.35.

¹H NMR [(CD₃)₂SO], δ(ppm): 0.71-0.77 (t, 6H), 1.38-1.47 (m, 4H),3.80-3.85 (t, 4H), 4.00 (s, 4H), 4.13 (s, 4H), 4.20 (s, 4H), 6.70-6.96(m, 8H).

Elemental. C₂₈H₃₆O₁₀N₂. Calculated: C, 60.00%; H, 6.43%; N, 5.00%;Found: C, 60.25%; H, 6.77%, H, 5.08%.

Iso-propyl diester of BAPTA. Yield 80% (10.2 g.). White powder. M.p.181-182° C. TLC analysis. Silica gel 60 F₂₅₄ on aluminum sheet. Eluent:chloroform:methanol (65:30, v/v). For indication the chromatogram issprayed by the indicator spray and then is charred at 350-400° C. Thecomposition of indicator spray is 4-methoxybenzaldehyde (10 ml), ethanol(200 ml), 98% sulfuric acid (10 ml) and glacial acetic acid (2 ml). Onespot. R_(f) 0.72.

¹H NMR [(CD₃)₂SO], δ (ppm): 1.07-1.09 (d, 12H), 4.00 (s, 4H), 4.08 (s,4H), 4.22 (s, 4H), 4.78-4.85 (m, 2H), 6.71-6.98 (m, 8H).

Elemental analysis. C₂₈H₃₆O₁₀N₂. Calculated: C, 60.00%; H, 6.43%; N,5.00%. Found: 59.78%; H, 6.50%; N, 5.00%.

Butyl diester of BAPTA. Yield 90% (12.1 g.). White powder. M.p. 183° C.TLC analysis. Conditions of analyses of diethyl and dibutyl esters ofBAPTA are analogous. One spot. R_(f) 0.42.

¹H NMR [(CD₃)₂SO]. δ ppm): 0.74-0.80 (t,6H), 1.09-1.18 (m, 4H),1.33-1.39 (m, 4H), 3.80-3.86 (t, 4H), 3.98 (s,4H), 4.10 (s, 4H), 4.17(s, 4H), 6.69-6.92 (m, 8H).

Elemental. C₃₀H₄₀O₁₀N₂. Calculated: C, 61.22%; H, 6.80%; N, 4.76%.Found: C, 61.54%; H, 7.10%, 5.03%.

Heptyl diester of BAPTA. Yield 70% (10.8 g.). White powder. M.p.146-147° C.

TLC analysis. Conditions of analysis of ethyl and heptyl diesters ofBAPTA are analogous. One spot. R_(f) 0.50.

¹H NMR [(CD₃)₂SO]. δ (ppm): 0.79-0.84 (t, 6H), 1.08-1.17 (broad s, 16H),1.34-1.43 (m, 4H), 3.79-3.87 (t, 4H), 3.98 (s, 4H), 4.13 (s, 4H), 4.17(s, 4H), 6.67-6.92 (m, 8H).

Elemental: C₃₆H₅₂O₁₀N₂. Calculated: C, 64.29%; H, 7.74%; N, 4.16%.Found: C, 64.37%; H, 7.82%; N, 3.88%.

Octyl diester of BAPTA. Yield 70% (11.3 g.). White powder. M.p. 155° C.TLC analysis. Conditions of analyses of diethyl and dioctyl esters ofBAPTA are analogous. One spot. R_(f) 0.55.

¹H NMR [(CD₃)₂SO], δ (ppm): 0.81-0.86 (t, 6H), 1.19-1.23 (broad s, 20H),1.29-1.34 (m, 4H), 3.83-3.87 (m, 4H), 3.98 (s, 4H), 4.11 (s, 4H), 4.19(s, 4H), 6.80-6.84 (m, 8H).

Elemental: C₃₈H₅₆O₁₀N₂. Calculated: C, 65.14%; H, 8.00%; N, 4.00%.Found: C, 64.91%; H, 8.20%; N, 3.76%.

Benzyl diester of BAPTA. Yield 70% (10.6 g.). White powder. M.p.161-163° C. TLC analysis. Conditions of analysis of ethyl and benzyldiester of BAPTA are analogous. One spot. R_(f) 0.64 (Benzyl diester isplotted on TLC plate in solution in dimethylformamide).

¹H NMR [(CD₃)₂SO], δ ppm): 4.02 (s, 4H), 4.18-4.19 (d, 8H), 4.97 (s,4H), 6.73-6.94 (m, 8H), 7.22-7.32 (m, 10H).

Elemental analysis. C₃₆H₃₆O₁₀N₂. Calculated: C, 65.85%; H, 5.49%; N,4.27%. Found: 65.56%, 5.83%, N; 4.12%.

2-(Dimethylamino)ethyl diester of BAPTA. Yield 70% (9.95 g.). Whitepowder.

M.p. 126-127° C. TLC analysis. Silica gel 60 F₂₅₄ on aluminum sheet.Eluent: chloroform:methanol:water 60:40:2 v/v. One spot. R_(f) 0.2.

¹H NMR (CDCl₃), δ (ppm): 2.57 (s, 12H), 2.60-2.63 (t, 4H), 3.60 (s, 4H),3.75-3.78 (t, 4H), 4.06 (s, 4H), 0.11 (s, 4H), 6.68-6.85 (m, 8H).

Elemental analysis. C₃₀H₄₂O₁₀N₄. Calculated: C, 58.25%; H, 6.80%; N,9.06%. Found: C, 57.94%; H, 6.90%; N, 8.97%.

Step 3a. Preparation of Sodium Salts of Diesters of BAPTA

Corresponding alkyl diester of BAPTA (0.019 Mol) is introduced into anErlenmeyer flask (500 ml), equipped with a magnetic stirrer. About 250ml of a mixture of methanol with water (1:1 v/v) is added to the ester.This mixture is vigorously stirred, because the ester is not dissolvedin the solution. A concentrated solution of NaHCO₃ (0.038 mol, 3.19 g.)or concentrated solution of MeONa (0.038 mol) in water is added to thestirring mixture, and after 5-8 hours the mixture becomes transparent.This indicates that the alkyl diester is converted into disodium salt.Methanol and water are evaporated under vacuum. The obtained salt isdried by azeotropic distillation with ethanol and diethyl ether.Finally, the salt is dried under vacuum (5-6 mm Hg) for 8 hours.

Ethyl diester of BAPTA, disodium salt. White powder. Yield 95% (10.4g.).

Elemental analysis. C₂₆H₃₀O₁₀N₂Na₂. Calculated: C, 54.16%; H, 5.21%; N,4.86%, Na, 7.98%. Found: 54.10%; H, 5.27%; N, 4.65%; Na, 8.10%.

Propyl diester of BAPTA, disodium salt. White powder. Yield 95% (10.9g.).

Elemental analysis. C₂₈H₃₆O₁₀N₂Na₂. Calculated: 55.63%; H, 5.63%; N,4.63%; Na, 7.61%. Found: 54.76%; H, 6.13%; N, 4.46%; Na, 6.73%.

Butyl diester of BAPTA, disodium salt. White powder. Yield 95% (11.2g.).

Elemental analysis. C₃₀H₃₈O₁₀N₂Na₂. Calculated: C; 56.96%, H; 6.01%, N;4.43%, Na; 7.28%. Found: C; 56.50%, H; 6.00%, N; 4.20%, Na; 7.30%.

Heptyl diester of BAPTA, disodium salt. White powder. Yield 90% (10.3g.).

Elemental analysis. C₃₆H₅₀O₁₀N₂Na₂. Calculated: C, 60.33%; H, 6.98%; N,3.91%; Na, 6.42%. Found: C, 59.88%; H, 7.49%; N, 4.12%; Na, 6.76%.

Octyl diester of BAPTA, disodium salt. White powder. Yield 90% (15.7g.).

Elemental analysis. C₃₈H₅₄O₁₀N₂Na₂. Calculated: C, 61.29%; H, 7.26%; N,3.76%; Na, 6.16%. Found: C, 60.90%; H, 7.81%; N, 3.26%; Na, 6.52%.

Step 3b. Preparation of Calcium Salts of Diesters of BAPTA

The corresponding diester of BAPTA (1 g.) is dissolved in 1 L mixture ofethanol with water (70:30 v/v). The equivalent molal of Ca(OH)₂ is addedto this solution. The obtained mixture is stirred by magnetic stirrer atroom temperature for 24 hours. Then for the salts of ethyl, propyl andbutyl diesters of BAPTA the solution is filtrated through Whatmann paperN1 and evaporated under vacuum (20-30 mm Hg) to dry. The precipitate iswashed three times by diethyl ether (each portion is 100 ml) and driedunder vacuum (2-3 mm Hg) at room temperature for 6 hours. For thecalcium salts of heptyl and octyl diesters of BAPTA the ethanol solutionis evaporated to dry. The precipitate is dissolved in 0.8 L of ethanol.The obtained mixture is filtrated through Whatmann paper N1 and thenethanol is evaporated under vacuum (20-25 mm Hg). The precipitate iswashed three times by diethyl ether (each portion is 100 ml) and driedunder vacuum (2-3 mm Hg) at room temperature for 6-7 hours.

Ethyl diester of BAPTA, calcium salt. White powder. Yield 90% (0.96 g.).

C₂₆H₃₀N₂O₁₀Ca. Calculated: C, 54.70%; H, 5.26%; N, 4.91%; Ca, 7.01%.Found: C, 54.32%; H, 5.40%; N, 4.81%; Ca, 6.81%.

Propyl diester of BAPTA, calcium salt. White powder. Yield 90% (0.98g.).

C₂₈H₃₄N₂O₁₀Ca. Calculated: C, 56.19%; H, 5.68%; N, 4.68%; Ca, 6.69%.Found: C, 56.22%; H, 5.88%; N, 4.51%; Ca, 6.51%.

Butyl diester of BAPTA, calcium salt. White powder. Yield 90% (0.90 g.).

C₃₀H₃₈N₂O₁₀Ca. Calculated: C, 57.50%; H, 6.07%; N, 4.47%; Ca, 6.39%.Found: C, 57.18%; H, 6.24%; N, 4.28%; Ca, 6.11%.

Heptyl diester of BAPTA, calcium salt. White powder. Yield 80% (0.85g.).

C₃₆H₅₀N₂O₁₀Ca. Calculated: C, 61.71%; H, 7.14%; N, 4.00%; Ca, 5.71%.Found: C, 61.44%; H, 7.24%; N, 4.18%; Ca, 6.31%.

Octyl diester of BAPTA, calcium salt. White powder. Yield 80% (0.83 g.).

C₃₈H₅₄N₂O₁₀Ca. Calculated: C, 61.79%; H, 7.32%; N, 3.79%; Ca, 5.42%.Found: C, 61.94%; H, 7.14%; N, 4.00%, Ca, 5.31%.

Example 2 Synthesis of BAPTA Diesters of Alkyl or Alkylaryl Ether ofMono-, Di- and Triethylene Glycol and Salts Thereof

The procedure for synthesis of salts of alkyl or alkylaryl ethers ofethylene glycols is a four-step process similar to the procedure forpreparation of the salts of the alkyl diesters of BAPTA.

Step 1. Preparation of BAPTA Anhydride

This first step of obtaining a BAPTA anhydride is identical to step 1 inthe procedure for synthesizing the alkyl diesters of BAPTA as describedabove in Example 1.

Step 2. Synthesis of Monoalkyl Ethers of Mono-, Di- and TriethyleneGlycol

The synthesis of monoalkyl ethers of mono-, di- and triethylene glycolis carried out according to following scheme:H(OCH₂CH₂)_(m)OH+Na→H(OCH₂CH₂)_(m)ONa+½H₂H(OCH₂CH₂)_(m)ONa+C_(n)H_(2n+1)Br→H(OCH₂CH₂)_(m)OC_(n)H_(2n+1)+NaBr

For example, m=1-3, n=5-18

About 0.8-0.9 g. of sodium (cut into small pieces where the diameter ofeach piece is 5-8 mm) are introduced, under argon atmosphere, into adouble-neck round-bottom flask (250 ml), equipped with a reversecondenser and magnetic stirrer. Ethylene glycol (35 ml, 0.62 Mol) isadded to the sodium, also under argon, and the flask is heated in oilbath at 70° C. with vigorous stirring. When most of the sodium isdissolved the rest of the sodium (typical quantity of sodium is 3.9 g.,0.17 Mol) is added piece by piece to the reaction mixture. It should benoted that sodium dissolution is accompanied by an increase in thetemperature of the reaction mixture together with the increased reactionrate. In order to avoid explosion, it is necessary to add sodium slowlyso that the reaction is well controlled. After all of the sodium isdissolved a drop funnel with the solution of the corresponding alkylbromide (21.5 g., 0.12 Mol) in tetrahydrofuran (60 ml) is added to thereaction flask. The solution from the drop funnel is introduceddrop-by-drop into the reaction flask. The temperature of the reactionmixture is kept at 70° C. Almost at once the precipitate of sodiumbromide appears and increases in quantity in the course of the reaction.After 16 hours the reaction mixture is cooled to room temperature andabout 150 ml of water is added to the organic solution. The product isextracted by two portions (40 ml each) of ethyl acetate. The combinedethyl acetate solution is washed with water and dried by sodium sulfate.The yellow solution of the product in ethyl acetate is discolored byheating with activated carbon. The colorless solution is separated fromthe carbon by filtration and the solvent is evaporated. The obtainedproduct is distilled under vacuum and analyzed for its physical andchemical characteristics.

Monoheptyl ether of ethylene glycol. Colorless liquid. B.p. 95° C./1 mmHg. Yield 70% (13.4 g.).

TLC analysis. Silica gel 60 F₂₅₄ on aluminum sheet. Eluent: ethylacetate:n-hexane, 2:1 v/v. Indicator: 4-methoxybenzaldehyde (10 ml),ethanol (200 ml), 98% sulfuric acid (10 ml) and glacial acetic acid (2ml). For indication the chromatogram is sprayed by the indicator sprayand then it is charred at 350° C. One spot. R_(f) 0.8.

¹H NMR (CDCl₃), δ (ppm): 0.84-0.90 (t, 3H), 1.27-1.33 (broad s, 8H),1.55-1.61 (m, 2H), 2.25-2.30 (t, 1H, signal of OH-group, its positionvariable), 3.43-3.54 (m, 4H), 3.69-3.75 (m, 2H).

Heptyl ether of diethylene glycol. Colorless liquid. B.p. 100° C./1 mmHg. Yield 70% (17.1 g.).

TLC analysis. Conditions of analyses of heptyl ether of mono- anddiethylene glycol are analogous. One spot. R_(f) 0.4.

¹H NMR (CDCl₃), δ (ppm): 0.84-0.90 (t, 3H), 1.27-1.32 (broad s, 8H),1.55-1.61 (m, 2H), 2,71(t, 1H, signal of OH-group), 3.45-3.48 (t, 2H),3.58-3.75 (m, 8H).

Heptyl ether of triethylene glycol. Colorless liquid. B.p. 107° C./1 mmHg. Yield 70% (20.8 g.).

TLC analysis. Conditions of analyses of monoheptyl ether of mono- andtriethylene glycol are analogous. One spot. R_(f) 0.3.

¹H NMR (CDCl₃), δ (ppm): 0.84-0.90 (t,3H), 1.26-1.29 (broad s, 8H),1.54-1.57 (m, 2H), 2.72 (t, 1H, signal of OH-group), 3.41-3.47 (t,2H),3.58-3.74 (m, 12H).

Octyl monoethylene glycol. Colorless liquid. B.p. 60° C./0.5 mm Hg.Yield 85%.

TLC analysis. Conditions of analyses of dioctyl ether of ethylene glycolare the same as above. One spot. R_(f) 0.7.

¹H NMR (CDCl₃), δ (ppm): 0.83-0.89 (t,3H), 1.25-1.27 (broad s, 10H),1.54-1.57 (m, 2H), 2.39 (t, 1H), 3.41-3.52 (m,4H), 3.67-3.73 (m, 4H).

2-Benzyloxyethanol, 2-Dodecyloxyethanol, 2-(2-Dodecyloxyethoxy)-ethanoland 2-[2-(2-Dodecyloxyethoxy)-ethoxy]-ethanol were purchased from FlukaCo.

Step 3. Synthesis of BAPTA Diesters of Monoalkyl Ethers of Mono-, Di-and Triethylene Glycol

The BAPTA anhydride of step 1 (1.5 g., 0.0034 Mol) and the correspondingmonoalkyl ether of mono-, di- or triethylene glycol of step 2 (10-12 ml)are introduced, under argon atmosphere, into a round-bottom single-neckflask (50 ml), equipped with a reverse condenser and a magnetic stirrer.The mixture is heated in an oil bath at 115-120° C. with vigorousstirring. After 1-1.5 hours the mixture becomes transparent. Heating iscontinued for another 1.5 hours, till the reaction is completed. Theflask is then cooled to room temperature and about 100 ml of petroleumether (b.p. 60-80° C.) is added. The formed precipitate is extracted bycentrifugation and washed three times with petroleum ether (40 ml eachwash). The solid product is dried under vacuum for 5 hours and analyzedto verify the product characteristics, as exemplified for the followingcompounds:

BAPTA diester of methylethylene glycol. White solid M.p. 151-152° C.Yield 90% (1.81 g.).

TLC analysis. Silica gel 60 F₂₅₄ on aluminum sheet. Eluent ischloroform:methanol (1:1 v/v). For indication the chromatogram issprayed by the indicator spray and then is charred at 100-150° C.Composition of indicator spray is 4-methoxybenzaldehyde (10 ml), ethanol(200 ml), 98% sulfuric acid (10 ml) and glacial acetic acid (2 ml). Onespot. R_(f) 0.14.

¹H NMR(CD₃OD), δ (ppm): 3.33 (s, 6H), 3.47-3.51 (t, 4H), 3.66 (s, 4H),3.85 (s, 4H), 4.02-4.06 (t, 4H), 4.35 (s, 4H), 7.02-7.11 (m, 8H).

Elemental analysis. C₂₈H₃₆O₁₂N₂. Calculated: C, 56.76%; H, 6.08%; N,4.73%. Found: C, 56.38%; H, 6.39%; N, 4.72%.

BAPTA diester of heptylethylene glycol. White solid. M.p. 111-112° C.Yield 90% (2.32 g.).

TLC analysis. Conditions of TLC analysis of BAPTA diester ofmethylethylene glycol and BAPTA diester of heptylethylene glycol are thesame. One spot. R_(f) 0.4.

¹H NMR [(CD₃)₂SO], δ (ppm): 0.81-0.86 (t,6H), 1.22 (broad s, 16H), 1,42(m, 4h), 3.27-3.32 (m, 4H), 3.37-3.40 (m, 4H), 3.96-3.99 (m, 8H), 4.12(s, 2H), 4.19 (s, 2H), 6.73-6.92 (m, 8H).

Elemental analyses. C₄₀H₆₀O₁₂N₂. Calculated: C; 63.16%, H; 7.90%, N;3.68%. Found: C; 63.30%, H; 8.44%, N; 3.76%

BAPTA diester of octylethylene glycol. White solid. M.p. 121-122° C.,Yield 80% (1.4 gr).

TLC analysis. Silica gel 60 on aluminum sheet. Eluent ischloroform:methanol (1:1, v/v). For indication the chromatogram issprayed by the the indicator spray and then is charred at 100-150° C.Composition of indicator spray is 4-methoxybenzaldehyde (10 ml), ethanol(200 ml), 98% sulfuric acid (10 ml), and glacial acetic acid (2 ml). Onespot. R_(f) 0.45.

¹H NMR (CDCl₃), δ (ppm) 0,84-0.89 (t, 6H), 1.26 (broad s, 20H),1.51-1.57 (m, 4H), 3.37-3.42 (t, 4H), 3.53-3.56 (m, 4H), 3.96 (s, 4H),4.03 (s, 4H), 4.17-4.21 (m, 4H), 4.37 (s, 4H), 6.87-6.94 (m, 4H),7.03-7.09 (m,4H).

Elemental analysis. C₄₂H₆₄N₂O₁₂. Calculated: C, 63.96%; H, 8.12%; N,3.55%. Found: C, 63.57%; H, 8.11%; N, 3.53%.

BAPTA diester of heptyldiethylene glycol. White solid. M.p. 95-96° C.Yield 85% (2.5 g.).

TLC analysis. Conditions of analysis of BAPTA diester of methylethyleneand BAPTA diester heptyldiethylene glycol are the same. One spot R_(f)0.40.

¹H NMR [(CD₃)₂SO], δ (ppm): 0.81-0.86 (t 6H), 1.23 (broad s, 16H), 1.45(m, 4H), 3.30-3.35 (m, 8H), 3.40-3.46 (m, 12H), 3.97-3.99 (m, 8H), 4.13(s, 4H), 4.19 (s, 4H), 6.74-6.92 (m, 8H), 12.37 (s, 2H).

Elemental. C₄₄H₆₈O₁₄N₂. Calculated: C, 62.26%; H, 8.02%; N, 3.30%.Found: C, 6.47%; H, 8.42%; N, 3.40%.

BAPTA diester of heptyltriethylene glycol. White solid. M.p. 63-65° C.Yield 85% (2.7 g.).

TLC analysis. Conditions of analysis of BAPTA diester ofheptyltriethylene glycol and BAPTA diester of methylethylene glycol arethe same. One spot. R_(f) 0.40.

¹H NMR [(CD₃)₂SO], δ (ppm): 0.81-0.87 (t, 6H), 1.23 (broad s, 16H), 1.45(m, 4H), 3.31-3.36 (m, 4H), 3.42-3.48 (m, 20H), 3.97-3.99 (m,8H), 4.13(s, 4H), 4.19 (s, 4H), 6.74-6.92 (m, 8H), 12.38 (s, 2H).

BAPTA diester of 2-benzyloxyethanol. White solid. Yield 80% (2.02 g.).

TLC analysis. Conditions of TLC analysis of BAPTA diester of2-benzyloxyethanol and BAPTA diester of heptylethylene glycol are thesame. One spot. R_(f) 0.5.

¹H NMR [(CD₃)₂SO], δ (ppm): 3.43-3.47 (m, 4H), 3.97 (s, 4H), 4.00-4.03(m, 4H), 4.12-4.16 (d, 8H), 4.40 (s, 4H), 6.70-6.93 (m, 8H), 7.23-7.30(m, 10H).

MS(APCI, ammonium acetate), MH⁺ 746.1 (Calculated for MH⁺ 745.8).

BAPTA diester of 2-dodecyloxyethanol. White solid. Yield 80% (2.44 g.).

TLC analysis. Conditions of TLC analysis of BAPTA diester of2-dodecyloxyethanol and BAPTA diester of heptylethylene glycol are thesame. One spot. R_(f) 0.5.

¹H NMR (CD₃OD), δ (ppm): 0.86-0.90 (t,6H), 1.27 (broad s, 36H), 150-1.56(m, 4h), 3.39-3.44 (t, 4H), 3.50-3.53 (m, 4H), 3.65 (s, 4H), 3.85 (s,4H), 4.01-4.05 (m, 4H), 4.33 (s, 4H), 6.87-7.11 (m, 8H).

MS (APCI, ammonium acetate), MH⁺ (902.1), calculated MH⁺ 902.2.

BAPTA diester of 2-(2-diodecyloxyethoxy)-ethanol. White solid. Yield 80%(2.68 g.).

TLC analysis. Conditions of TLC analysis of BAPTA diester of2-(2-dodecyloxyethoxy)-ethanol and BAPTA diester of heptylethyleneglycol are the same. One spot. R_(f) 0.5.

¹H NMR (CD₃OD), δ (ppm): 0.88-0.93 (t, 6H), 1.28 (broad s, 36H),1.51-1.60 (m, 4h), 3.44-3.49 (t, 4H), 3.54-3.61 (m, 12H), 3.66 (s, 4H),3.84 (s, 4H), 4.04-4.07 (m, 4H), 4.35 (s, 4H), 6.68-7.13 (m, 8H).

MS(APCI, ammonium acetate) MH⁺ (989.7), calculated MH⁺ 990.2

BAPTA diester of 2-[2-(2-dodecyloxyethoxy)-ethoxy]-ethanol. White solid.Yield 70% (2.32 g.).

TLC analysis. Conditions of TLC analysis of BAPTA diester of2-[2-(2-dodecyloxyethoxy)-ethoxy]-ethanol and BAPTA diester ofheptylethylene glycol are the same. One spot. R_(f) 0.5.

¹H NMR (CD₃OD), δ (ppm): 0.87-0.93(t, 6H), 1.29 (broad s, 36H),1,52-1.57 (m, 4h), 3.42-3.47 (t, 4H), 3.53-3.74 (m, 24H), 3.83 (s, 4H),4.03-4.07 (m, 4H), 4.36 (s, 4H), 6.91-7.13 (m, 8H).

MS (APCI, ammonium acetate), MH⁺ (1078.8), calculated MH⁺ 1078.4.

Step 4a. Preparation of Disodium Salt of BAPTA Diesters of MonoalkylEthers of Mono-, Di- or Triethylene Glycol.

The corresponding BAPTA diester of monoalkyl ether of mono-, di- ortriethylene glycol (0.0025 Mol) is dissolved in methanol (arround 10 mlof alcohol is necessary for dissolving 1.0 g. of BAPTA diester) and theobtained solution is introduced into an Erlenmeyer flask (50 ml),equipped with a magnetic stirrer. A water solution of sodium bicarbonate(0.005 Mol in 2 ml) is added to a methanol solution of the BAPTA diesterand the mixture is stirred for 2 hours at room temperature. The solventis then evaporated under vacuum (30 mm Hg). The obtained precipitate isdried three times by azeotropic distillation with ethanol and two timeswith diethyl ether. Finally, the obtained product is washed with hexaneand is dried under vacuum.

BAPTA diester of methylmonoethylene glycol, disodium salt. White solid.

Hygroscopic. Yield 95% (1.5 g.).

Elemental analysis. C₂₈H₃₄O₁₂N₂Na₂. Calculated: C, 52.80%; H, 5.35%; N,4.40%; Na, 7.23%. Found: 52.20%; H, 5.59%; N, 4.49%; Na, 7.30%.

BAPTA diester of heptylmonoethylene glycol, disodium salt. White solid.

Hygroscopic. Yield 95% (1.9 g.).

Elemental analysis. C₄₀H₅₈O₁₂N₂Na₂. Calculated: C, 59.70%; H, 7.21%; N,3.48%; Na, 5.72%. Found: C, 59.60%; N, 7.75%; N, 3.51%; Na, 5.51%.

BAPTA diester of heptyldiethylene glycol, disodium salt. White solid.

Hygroscopic. Yield 95% (2.1 g.).

Elemental analysis. C₄₄H₆₆O₁₄N₂Na₂. Calculated: C, 59.19%; H, 7.40%; N,3.14%; Na, 5.16%. Found: C, 58.55%; H, 7.43%; N, 3.46%; Na, 5.49%.

BAPTA diester of heptyltriethylene glycol, disodium salt. White wax.Very hygroscopic. Yield 90% (2.2 g.).

Elemental analysis. C₄₈H₇₄O₁₆N₂Na₂. Calculated: C, 58.77%; H; 7.55%; N,2.86%; Na, 4.69%. Found: C, 57.98%; H, 8.03%; N, 2.94%; Na, 4.64%.

BAPTA diester of octylethylene glycol, disodium salt. White solid. Yield80%.

TLC analysis. Silica gel 60 on aluminum sheet. Eluent ischloroform:methanol (1:1, v/v). For indication the chromatogram issprayed by the the indicator spray and then is charred at 100-150° C.Composition of indicator spray is 4-methoxybenzaldehyde (10 ml), ethanol(200 ml), 98% sulfuric acid (10 ml), and glacial acetic acid (2 ml). Onespot. R_(f) 0.45.

¹H NMR (CDCl₃), δ (ppm) 0,84-0.89 (t, 6H), 1.26 (broad s, 20H),1.51-1.57 (m, 4H), 3.37-3.42 (t, 4H), 3.53-3.56 (m, 4H), 3.96 (s, 4H),4.03 (s, 4H), 4.17-4.21 (m, 4H), 4.37 (s, 4H), 6.87-6.94 (m, 4H),7.03-7.09 (m,4H).

Elemental analysis. C₄₂H₆₄N₂O₁₂. Calculated: C, 63.96%; H, 8.12%; N,3.55%. Found: C, 63.57%; H, 8.11%; N, 3.53%.

BAPTA diester of 2-benzyloxyethanol, disodium salt. White solid.Hygroscopic.

Yield 90% (1.92 g.). Water content is 7.0%

Elemental analysis. C₄₀H₄₂N₂O₁₂Na₂.3H₂O. Calculated: C, 57.01%; H;5.70%; N, 3.33%; Na, 5.46%. Found: 56.44%; H, 5.90%; N, 3.49%; Na,5.50%.

BAPTA diester of 2-dodecyloxyethanol, disodium salt. White solid.Hygroscopic.

Yield 90% (2.3 g.). Water content is 2.8%.

Elemental analysis. C₂₈H₃₄N₂O₁₂Na₂.1.5H₂O Calculated: C, 61.73%; H,8.33%; N, 2.88%; Na, 4.73%. Found: 61.34%; H, 8.45%; N, 2.76%; Na,4.99%.

BAPTA diester of 2-(2-dodecyloxyethoxy)-ethanol, disodium salt. Whitesolid.

Hygroscopic. Yield 85% (2.35 g.). Water content is 4.3%.

Elemental analysis. C₅₄H₈₆N₂O₁₄Na₂.2.5H₂O Calculated: C, 60.15%; H,8.51%; N, 2.60%; Na, 4.27%. Found: 59.68%; H, 8.33%; N, 2.46%; Na,4.75%.

BAPTA diester of 2-[2-(2-dodecyloxyethoxy)-ethoxy]-ethanol, disodiumsalt.

White solid. Hygroscopic. Yield 85% (1.5 g.). Water content is 2.3%

Elemental analysis. C₅₈H₉₄N₂O₁₆Na₂.1.5H₂O Calculated: C, 60.66%; H;8.45%; N, 2.44%; Na, 4.00%. Found: 60.20%; H, 8.32%; N, 2.32%; Na,4.30%.

Step 4b. Preparation of Calcium Salt of BAPTA Diesters of MonoalkylEthers of Mono-, Di- or Triethylene Glycol.

The corresponding monoalkyl ether of mono-, di- or triethylene glycoldiester of BAPTA (0.0025 Mol) is dissolved into 250 ml methanol. About3-5 ml of water is added to this solution. The obtained solution isintroduced into an Erlenmeyer flask (300 ml), equipped with a magneticstirrer. The powder of CaH₂ (0.0025 Mol) is added to this solution withvigorous stirring. The stirring is continued for 3 hours at roomtemperature. After 3 hours the mixture is filtered through paper filter(Whatman N1) and the obtained solution is evaporated under vacuum (10-15mm Hg). The precipitate is dried three times by azeotropic distillationwith ethanol (each portion is 25-30 ml) and two times with diethylether. Finally, the product is washed with hexane and it is dried undervacuum (5 mm Hg) for 5 hours at room temperature.

Methylmonoethylene glycol diester of BAPTA, calcium salt. White powderYield 90% (1.42 g.). C₂₈H₃₄N₂O₁₂Ca. Calculated: C, 53.33%; H, 5.40%; N,4.44%; Ca, 6.35%. Found: C, 53.74%; H, 5.78%; N, 4.43%; Ca, 5.90%.

Heptylmonoethylene glycol diester of BAPTA, calcium salt. White powder.Yield 90% (1.79 g.). C₄₀H₅₈N₂O₁₂Ca. Calculated: C, 60.15%, H, 7.27%; N,3.51%; Ca, 5.01%. Found: C, 60.32%, H, 7.63%, N, 3.54%; Ca, 4.59%.

Octylmonoethylene glycol diester of BAPTA, calcium salt White powder.Yield 90% (1.81 g.). C₄₂H₆₂N₂O₁₂Ca. Calculated: C, 61.01%; H, 7.50%; N,3.38%; Ca, 4.84%. Found: C, 61.00%; H, 7.82%; N, 3.54%; Ca, 4.88%.

Heptyldiethylene glycol diester of BAPTA, calcium salt. White solid.Yield 80% (1.77 g.). C₄₄H₆₆N₂O₁₄Ca. Calculated: C, 59.59%; H, 7.44%; N,3.16%; Ca, 4.51%; Found: C, 59.61%; H, 7.79%; N, 3.15%; Ca, 4.04%.

Methyltriethylene glycol diester of BAPTA, calcium salt. White solid.Yield 80% (1.61 g.). C₃₆H₅₀N₂O₁₆Ca. Calculated: C, 53.60%; H, 6.20%; N,3.47%; Ca, 4.96%; Found: C, 53.95%; H, 6.33%; N, 3.20%; Ca, 4.73%.

Example 3 DP-BAPTA Reduces the Basal Activity as well as theTNFα-Induced Activity of MMP-9 in C6-Glioma Cells

The effect of DP-b99 on MMP-9 activity was tested on cultured C6 gliomacells either in the absence (basal activity) or following treatment withTumor Necrosis Factor alpha (TNFα) to induce gelatinases.

C6 rat glioma cells (ATCC; CRL-2199) grown on 100 mm petri dishes weredetached by trypsinization and cultured to a density of 10⁵ cells/wellon 24-well plates in DMEM+10% FCS. The TNFα-treated cells which werestimulated to induce gelatinases were incubated, on the next day afterplating, for 18 hours in DMEM without serum in the presence of either 20ng/ml or 40 ng/ml

TNFα (R&D, cat. #410-TRNC). Different concentrations of the testedcompound, as indicated, in 0.48% fatty acid free BSA, 0.4% ethanol wereadded to the cells that were then incubated for a total of 18-24 hoursat 37° C. Cells treated with vehicle only served as control group.

Conditioned media (CM) were then collected, centrifuged at 2000 rpm for5 min., and supernatants were transferred to new tubes. MMP gelatinaseactivity was determined by using zymogram gels (Invitrogen, cat.#EC6175) which include gelatin as a substrate. Samples were loaded ongels in 62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS and traces ofbromophenol-blue. Gels were run in Tris/glycine-SDS running buffer,washed for 30 min. with renaturing buffer (Invitrogen) and for further30 min. with developing buffer (Invitrogen). MMP activity was developedovernight at 37° C. with fresh developing buffer. The gels were thenstained for one hour with Coomasie Blue (Brilliant Blue R, Sigma, cat.#B-8647) in 40% methanol and 7% acetic acid, and destained with 30%methanol/10% acetic acid. Digested gelatin areas formed by the MMPs(showed as clear bands) were photographed using Kodak digital Science™Image station. Band analysis was determined using the Kodak digitalScience™ 1D Image Analysis Software. Treatments were performed intriplicates and were subjected to three separate zymogram gels.

It was found that treatment with TNFα caused a 3- to 10-fold increase inMMP-9 activity. Added DP-BAPTAs (20 μM) inhibited both basal andTNFα-induced MMP activity in C6-glioma cells. Percentage inhibition onthe basal and TNFα-induced MMP-9 activity was around 25% and up to 60%,respectively.

Example 4 DP-BAPTA Reduces Expression/Release of TNFα-Induced MMP-9 andInhibits Enzyme Activity

The effect of DP-b 109 on total amount of MMP-9 and its activity wastested in a human glioma cell line following treatment with TumorNecrosis Factor alpha (TNFα).

A-172 human glioma cells (ATCC; CRL-1620) were grown and treated with 10ng/ml TNFα in the presence of 0, 20 or 50 μM DP-b109 following theprocedure as described above in Example 3. Cells treated with vehiclewithout the TNFα treatment, served as control groups.

CM from each treatment group was collected and subjected to zymogramgels (see procedure in Example 3), to MMP-9 immunoassay and to MMP-9activity assay.

The total amount of MMP-9 protein was determined by immunoassay usingthe Quantikine™ (R&D Systems, U.S.A., Cat # DMP900) kit and followingthe manufacturer's instructions.

MMP-9 activity was assayed using the MMP-9 activity assay system(BioTrak™, Amersham, U.K., Cat. # RPN 2634), which quantifies the amountof the active MMP-9 form (see BioTrak kit instructions).

In FIG. 1A are shown the results of the immunoassay, where it isdemonstrated that the amount of total MMP-9 is increased about 10-foldsin the presence of TNFα. FIG. 1A demonstrates that DP-b109 reduces, in adose-dependent fashion, the level of MMP-9 protein measured in thecollected conditioned media. This reduction may be attributed todecrease in MMP-9 expression, to release of the enzyme or to combinationof both.

As can be seen in FIG. 1B, in the presence of TNFα there was a 35%-40%increase in active MMP-9. In the presence of the added DP-b 109, thisincrease was reduced to the baseline level, i.e. to the level of MMP-9activity measured in the absence of the TNFα-induction.

Conclusions:

It was shown that the DP-BAPTA inhibitory effect on MMP-9 activity is intwo levels: a) reduction of protein expression/release, and b)inhibition of MMP-9 enzymatic activity.

Example 5 DP-BAPTA Inhibits MMP-9 Activity Induced in C6-Glioma Cells byEither TNFα or PMA

The effect of DP-BAPTA on MMP-9 activity induced with either TNFα orPhorbol 12-myristate 13-acetate (PMA) was tested in C6 glioma cells.

C6 glioma cells were grown and treated as described above in Example 3,except that cells were stimulated with either 20 ng/ml TNFα (R&D, cat.#410-TRNC) or 0.1 μM Phorbol 12-myristate 13-acetate (PMA, Sigma, cat.#P-8139).

It was shown that both TNFα and PMA increased MMP-9 secretion by about10 fold. The results of an experiment where the effect of differentconcentrations of DP-109 was tested are shown in FIG. 2.

As can be seen in FIG. 2, DP-109 decreased the MMP-9 activity induced byeither TNFα or PMA in a dose dependent fashion. The calculated IC₅₀ wassimilar for both treatments, IC₅₀=˜10 μM.

These results obtained with two different stimulators of MMP-9; theknown inducer of MMP-9 activity, TNFα, and the protein kinase C (PKC)activator, PMA, suggest that DP-BAPTA inhibitory effect on MMP-9 is notthrough an interaction with the TNFα receptor, but downstream to it,either by blocking the expression of MMP-9, or by direct inhibition ofthe MMP-9 enzyme activity.

Example 6 Effect of DP-BAPTA on MMP-9 Activity in Comparison to BAPTAand BAPTA-AM

Several DP-BAPTA molecules were screened for their effect on MMP-9activity and were compared to the effect of the related chelatingcompounds 1,2-bis(2 aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA) and its lipophilic analog1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid acetoxymethylester (BAPTA-AM).

C6 glioma cells were plated and treated with 20 ng/ml TNFα following theprotocol described above in Example 3, either in the presence or absenceof a tested compound as indicated. Each of the tested DP-BAPTAs, BAPTAor BAPTA-AM was added at the following final concentrations: 1, 5, 10and 20 μM. As controls served cells treated with vehicle only.

Assays were performed in triplicates and zymogram analysis was carriedout as described above in Example 3. IC₅₀ values for each of the testedDP-BAPTA compounds were calculated. The results are summarized in Table1.

TABLE 1 Inhibitory effect on TNFα-induced MMP-9 activity Tested compoundR in formula I IC₅₀(μM) vehicle — — DP-b99 C₈H₁₇OCH₂CH₂ ~40 DP-b109C₁₈H₃₇OCH₂CH₂ 10–20 DP- b460 C₁₂H₂₅OCH₂CH₂ 20 DP-b458 C₁₂H₂₅(OCH₂CH₂)₂n.d. DP-b440 benzyl-OCH₂CH₂ 12 BAPTA H No inhibition BAPTA-AMtetra-ester (acetoxymethyl) Increased MMP9 activity n.d.—not determinedConclusions:

The most potent inhibitors among the tested compounds were1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-benzyloxyethyl acetate),N,N′-acetic acid, disodium salt [DP-b440] and1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-octodecyloxyethyl acetate),N,N′-diacetic acid, disodium salt [DP-b109] with calculated IC₅₀ of 12μM and 10-20 μM, respectively.

It is important to note that the parent chelating compound, BAPTA, didnot affect the MMP-9 activity. The related tetra-ester BAPTA analog,BAPTA-AM, increased rather than decreased the MMP-9 activity in the testmodel system used (C6 glioma cells stimulated with TNFα).

Example 7 DP-BAPTA Inhibits MMP-9 Activity in Primary Cultured GlialCells

The effect of DP-BAPTA molecules on un-stimulated MMP-9 (basal activity)and their ability to inhibit MMP-9 activation induced by TNFα, wastested in primary glial cells.

Cortical glial cells from rat embryos (day 18 of pregnancy) were seededon poly-D-lysine coated 24-well plates with MEM medium containing 5%FCS, 5% HS, 2 mM 1-glutamine and 0.6% glucose, at a density of 5×10⁴cells/well. After 4 days in culture, the medium was changed to MEM+10%FCS. When glial cells reached confluence (around 17 days after plating),they were exposed for 24 hours to 10 ng/ml or 20 ng/ml human TNFα inorder to induce MMP-9. The induction was performed either in the absenceor presence of 25 μM DP-b99 (dilution 1:200 with medium from a stock of5 mM in sodium citrate buffer) in a serum-free medium containing 5 mg/mlBSA (essentially fatty acid free). Conditioned media (CM) were collectedand 12 μl of each of the triplicates was subjected to separate zymogramgels, as described above in Example 3.

A representative zymogram of MMP-9 activity in conditioned medium (CM)from control (un-stimulated) glial cells and from cultures treated with10 or 20 ng/ml TNFα, either with or without DP-b99, is depicted in FIG.3.

As can be seen in FIG. 3A, the two forms of MMP-9 were separated on thegel; the upper band represents the pro-MMP-9 with the higher molecularweight, while the lower band (lower MW) represents the biologicallyactive form of the enzyme.

A quantitative analysis of the MMP-9 bands, representing the pro- andactive forms of the enzyme, was performed on the triplicate zymograms.The results were normalized to percentage of control values (=100%) andare depicted in FIGS. 3B and 3C that represent, respectively, thebiologically active- and pro-MMP-9 forms of the enzyme.

The results clearly demonstrated that in the un-stimulated cells DP-b99increased the band intensity of pro-MMP-9 on the account of the activeMMP-9 band, which was abolished. In the TNFα treated cells both pro- andactive forms of MMP-9 were reduced.

Conclusions:

The DP-b99 inhibitory effect on basal MMP-9 activity in glial cells wasdemonstrated by showing an increase in band intensity of the Pro-MMP-9on the account of the active MMP-9 form. In TNFα-treated cells, DP-b99reduced both the pro- and active forms of the enzyme.

Example 8 Effect of DP-BAPTA on TNFα Release from Primary Glial Cells

The effect of various DP-BAPTA molecules on the levels of TNFα releasedin response to stimulation of primary glial cells withlipopolysaccharide (LPS) was measured.

Cortical rat glial cells were plated as described above in Example 7.When glial cells reached confluence (17 days after plating), they wereexposed for 18 hours to 0.5 μg/ml LPS (E. coli 0111:B4, Calbiochem, cat.#437627) in the absence or presence of 20 μM DP-b99, DP-b109, DP-b458,DP-b440, DP-b460 or DP-b464 (=1,2-bis(2-aminophenoxy)ethane,N,N′-di{2-[2-(2-dodecyloxyethoxy) ethoxy]-ethyl acetate}, N,N′-diaceticacid) in a serum free medium. CM were collected and analyzed for thepresence of TNFα by using an ELISA assay (DuoSet, R&D, cat. #DY510).

As can be seen in FIG. 4, LPS increased the release of TNFα by around 10folds over control levels. All DP-BAPTA tested compounds inhibited thisincrease.

Conclusions:

The various DP-BAPTA compounds were effective to a different degree intheir ability to reduce the induction of TNFα release in primary glialcells.

The results suggest a possible role for the DP-BAPTA molecules inblocking the activity of TNFα Converting Enzyme (TACE) in glial cells,hence implying a potential use of these molecules in inhibiting orinterfering with neuro-inflammatory processes.

Example 9 Effect of DP-BAPTA on TNFα Release from Macrophages

The effect of DP-BAPTA on the levels of TNFα released in response tostimulation with lipopolysaccharide (LPS) is also tested in macrophagecell lines. Mouse macrophage cells grown on 100 mm petri dishes, aredetached by scraping and re-cultured to a density of about 10⁵cells/well on 24-well plates in DMEM+10% FCS. 48 hours later, cells areexposed for 18 hours to 0.5 μg/ml LPS (E. coli 0111:B4, Calbiochem, cat.#437627) in serum free medium in the absence or presence of 20 μMDP-BAPTA. Conditioned media are collected for TNFα analysis which isperformed by using an ELISA assay (DuoSet, R&D, cat. #DY410).

The ability of various DP-BAPTA molecules to inhibit TNFα release frommacrophages, may indicate a potential use of these molecules also intreating or ameliorating diseases and conditions related to peripheralinflammatory processes.

Example 10 Effect of DP-b99 on MMP-9 Activation In-Vivo

In order to test the effect of DP-BAPTA on MMP-9 activity in-vivo, thefollowing model system for brain ischemia and the following protocolwere employed.

Six Sprague-Dawley (SD) rats were subjected to unilateral (righthemisphere) Middle Cerebral Artery Occlusion (MCAO) for 2 hours. 5 μg/kgDP-b99 in 0.02% sodium citrate in saline was i.p. administered to threeanimals in a single dose, followed by 2 hrs. reperfusion. The otherthree animals were treated with vehicle instead of DP-b99. A rat thatwas only operated with no further treatment was used as a sham control.

The seven rats were sacrificed after 24 hrs. and each hemisphere oftheir brains was subjected separately to lysis and extracted forenzymatic activity. Brains were minced and solubilized in lysis buffer(25 mM Tris-HCl pH 7.5, 1% IGEPAL CA-630—a non-ionic detergent fromSigma, 100 mM NaCl, 0.5 U/ml aprotinin, 0.01% sodium azide) at a finalconcentration of 400 mg/ml. The preparations were incubated for 24 hrs.at 4° C. before lysates were centrifuged at 14,000 rpm for 15 min. andsupernatants were collected.

Protein concentration was determined by the Bradford assay and 40 μgprotein from each lysate were loaded on gelatin zymogram gels fordetermination of MMP-9 levels. Gels were developed for 48 hrs.Quantitative analysis of the gels was performed using the Kodak DigitalSystem and Kodak 1D software as described in Example 3. The results areshown in FIG. 5.

Same level of MMP activity was measured in the two hemispheres of thesham-operated rat. In the MCAO-treated rats, as expected, induction ofMMP-9 activity was demonstrated in the right (R-injured) hemisphere,while no significant increase was observed in the left (L-intact)hemisphere.

As shown in FIG. 5, treatment with DP-b99 inhibited this induction inMMP activity. Calculated R/L ratio in the DP-b99 treated rats was 3.0 incomparison to R/L=4.9 in the vehicle treated rats.

It is important to note that in contrast to MMP-9 that was inhibited byDP-b99, the activity level of the other tested gelatinase, MMP-2, wasunaffected by the treatment with DP-b99.

Conclusions:

DP-b99 reduced the increase in MMP-9 activity induced by focal brainischemia in rats. The results of this in-vivo study correlate with thoseof the in vitro experiments (Examples 3 to 7) that showed that DP-BAPTAsreduce MMP-9 activity induced in C6 and A-172 glioma cells and inprimary glial cells. These findings indicate that DP-BAPTAs can inhibitMMP-9 activity in vivo, thus may be useful in interfering with damagingneuro-inflammatory processes.

Example 11 DP-b-99 Inhibits Calpain Activity in Primary Cortical Neurons

Calpain activity in primary cortical neurons was evaluated followingactivation of the enzyme by H₂O₂. Calpain activity was measured bymonitoring proteolytic degradation of the calpain substrate α-spectrin,from the 280 kDa full-length protein to the 150 kDa degradation product.

Culture Preparation: Primary cortical neurons were prepared from thebrains of embryonic day 16-17 (E16-17) rat fetuses. Cells from embryosof one mother were resuspended in 300 ml of “primary medium” (NBM Gibco,Glasgow, Scotland) with 0.5 mM glutamine, 0.4 units/ml penicillin, 0.4μg/ml streptomycin, and B27 supplement (Gibco, Glasgow, Scotland), plus25 μM glutamic acid. Cells (˜3×10⁵/ml) were seeded, 1 ml/well, in24-well plate for viability assay, or 4 ml/well in 6-well plate foranalysis of calpain activity. Every 3-4 days, half of the medium wasreplaced with fresh “primary medium”. Cells were used for experimentsafter 5-7 days.

Induction of Oxidative Stress: H₂O₂ was added at the indicatedconcentrations to cells in “primary medium” from a solution of 10 mMthat was prepared in PBS immediately before use. Viability wasdetermined after 18 to 24 hours. DP-b99 was added to the medium 1-2hours prior to induction of the oxidative stress.

Assay for Calpain activity: Primary cortical neurons in 6-well plateswere lysed in 100 μL RIPA buffer (50 mM Tris pH 7.5; 150 mM NaCl; 0.5%DOC; 1% Triton X-100; 0.1% SDS; 1 mM Nappi; 2 mM EDTA) plus proteaseinhibitors (Bohringer, Manheim, Germany). After 10 min. incubation onice, lysates were cleared by 15 min. centrifugation at 20,000 g at 4° C.Samples including 10-50 μg protein were separated on gradient 4-12%SDS-PAGE (Nu-PAGE, NOVEX, with MES buffer), and blotted tonitrocellulose paper according to the manufacture instructions. Todetect the various forms of α-spectrin, blots were reacted withanti-spectrin antibodies (Affinity FG6090 1:1000), followed byHRP-second antibodies (Santa Cruz, USA), followed by ECL reaction(Amersham, Buckinghamshire, UK). Detection of bands was performed usingthe Image Station 440 (Kodak Digital System). The non-cleaved spectrinran at ˜280 kDa, while the calpain cleaved form ran at ˜150 kDa.Quantitation of the bands was performed using the Kodak 1D software.

In the experimental system described above it was found thatcalpain-cleaved product appears after 2 hours and reaches a maximallevel at 4 hours following addition of H₂O₂.

The effect of DP-b99 on calpain activity was studied 4 hours afteraddition of H₂O₂ by monitoring the H₂O₂-induced cleavage of spectrin.The cortical primary cells were pre-treated with DP-b99 (15 μg/ml) orwith the commercially available calpain inhibitor MDL28170 (25 μM) for 1hour before H₂O₂ (50 μM) was added. Calpain activity was determined byusing the “Calpain activity” assay described above.

As can be seen in FIG. 6, DP-b99 inhibited calpain activity to a similarextent as the known calpain inhibitor MDL28170. Percent inhibition byDP-b99 was from 40% to 60% in different experiments.

Conclusions:

DP-b99 inhibits H₂O₂-induced calpain activity similarly to thecommercial calpain inhibitor MDL28170.

Example 12 DP-b-99 Inhibits Induced Spectrin Cleveage In Vivo

The effect of DP-b99 on calpain activity was further evaluated in vivoin transient focal cerebral ischemia model system in rats.

Transient middle cerebral artery (MCA) occlusion was performed in maleSprague Dawley (SD) rats by applying 4-0 silicone-coated nylonmonofilament through external carotid artery. The animals wereanesthetized with 4% halothane and maintained at 1.5% halothane in a 1:2mixture of nitrous oxide and oxygen without tracheotomy and allowed tobreathe spontaneously. Via a ventral cutaneous the common, rightexternal and internal carotid arteries (ECA) were exposed. A suture wasintroduced into a ligated right external carotid artery, in a retrogradefashion towards the carotid bifurcation. It was then directed distallyup to the right internal carotid artery to a distance of 20 mm from thecarotid bifurcation to permanently occlude the origin of the MCA. It wassecured with silk ligatures. Two hours after occlusion the monofilamentwas withdrawn to allow reperfusion and the cutaneous wound was suturedand cleaned. The whole procedure took about 25 min., with rectaltemperature maintained throughout at 37° C.

DP-b99 was intraperitoneally (i.p.) administered in a single dose (5μg/kg) at the start of reperfusion. MCAO-treated animals i.p.-injectedwith vehicle only served as control group. Four hours later, the animalswere sacrificed and their brains were cut into two hemispheres andimmediately frozen at −80° C.

Assay for Calpain activity: Each hemisphere was separately lysed byhomogenization on ice in 5 ml RIPA buffer (50 mM Tris pH 7.5; 150 mMNaCl; 0.5% DOC; 1% Triton X-100; 0.1% SDS; 1 mM Nappi; 2 mM EDTA) plusprotease inhibitors (Bohringer, Manheim, Germany). Lysates were clearedby 30 min centrifugation at 3,000 g at 4° C., followed by 30 min.centrifugation at 20,000 g at 4° C. Samples containing 50 μg proteinwere separated on gradient 4-12% SDS-PAGE (Nu-PAGE, NOVEX, with MESbuffer), and blotted to nitrocellulose paper according to themanufacture instructions. To detect the various forms of α-spectrin,blots were reacted with anti-spectrin antibodies (Affinity FG60901:1000), followed by HRP-second antibodies (Santa Cruz, USA), followedby ECL reaction (Amersham, Buckinghamshire, UK). Detection of bands wasperformed using the Image Station 440 (Kodak Digital System). The intactspectrin ran at ˜280 kDa, while the calpain-cleaved form ran at ˜150kDa. Quantitation of the bands was performed using the Kodak 1Dsoftware.

In the experimental system described above it was found that in animalssubjected to MCAO, the calpain-cleaved spectrin (˜150 kDa band)increased in the right (R, ischemic) hemisphere relative to the left (L,intact) hemisphere. In the sham-operated animals the amount ofcalpain-cleaved spectrin was similar in the two hemispheres.

The increase in the cleaved form of spectrin for each animal (n=2 foreach treatment), was presented as the ratio between the 150 kDa—spectrinbands in the right (ischemic) and left (intact) hemispheres. The resultsare summarized in Table 2.

TABLE 2 Inhibition of increase in calpain-cleaved spectrin by DP-b99Cleaved spectrin Treatment (R/L hemisphere) Sham 0.92 ischemia + vehicle2.96 ± 0.26 ischemia + DP-b99 1.42 ± 0.08

As shown in Table 2, the increase in calpain-cleaved spectrin in theright hemisphere relative to the left (R/L ratio) in the animalssubjected to the unilateral MCAO and treated with vehicle only, wasabout 3 folds higher than the ratio in the sham-operated animals. In theanimals treated with DP-b99, the increase in cleaved spectrin was muchreduced; only about 50% increase over the sham-operated values.

Conclusions:

DP-b99 inhibits the increase in calpain activity induced by ischemia invivo. Calpain inhibition by DP-b99 may be responsible, at least in part,for the neuroprotective effect exhibited by this molecule.

While the present invention has been particularly described, personsskilled in the art will appreciate that many variations andmodifications can be made.

Therefore, the invention is not to be construed as restricted to theparticularly described embodiments, rather the scope, spirit and conceptof the invention will be more readily understood by reference to theclaims which follow.

1. A method for treating or managing a metalloproteinase (MMP) orcalpain related disease or disorder in a mammal, the disease or disorderbeing glioma comprising administering to a mammal in need thereof, apharmaceutical composition comrising a therapeutically effective amountof a compound of the general formula (I):

wherein R is saturated or unsaturated alkyl, cycloalkyl, arylalkyl orcycloalkyl-alkyl radical having from 1 to 28 carbon atoms which may beinterrupted by any combination of 1-6 oxygen and/or nitrogen atoms,provided that no two oxygen atoms or an oxygen and a nitrogen atom aredirectly connected to each other; and M denotes a hydrogen or aphysiologically acceptable cation.
 2. The method according to claim 1wherein said method further comprises treating mammal with additionaltherapeutic treatment.
 3. The method according to claim 1, wherein saidmammal is a human.
 4. The method according to claim 1 wherein R in thecompound of Formula (I) is a phenylalkyl, and alky interrupted by zeroto three oxygen atoms, or a monoalkyl ether of mono-, di, ortri-ethylene glycol.
 5. The method according to claim 1, wherein R inthe compound of Formula (I) is selected from the group consisting of:C₈H₁₇, C₈H₁₇OCH₂CH₂, C₁₈H₃₇, C₁₈H₃₇OCH₂CH₂, benzyl-CH₂OCH₂CH₂,C₁₂H₂₅OCH₂CH₂, C₁₂H₂₅(OCH₂CH₂)₂ and C₁₂H₂₅(OCH₂CH₂)₃.
 6. The methodaccording to claim 1, wherein the metalloproteinase is MMP-9.
 7. Themethod according to claim 2, wherein said additional treatment isselected from the group consisting of chemotherapy, irradiation therapy,immunotherapy, genetic therapy and surgery.
 8. The method according toclaim 1, wherein said compound of Formula (I) is selected from the groupconsisting of: 1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-octoxyethylacetate), N,N′-diacetic acid; 1,2-bis(2-aminophenoxy)ethane,N,N′-di(2-octodecyloxyethyl acetate), N,N′-diacetic acid;1,2-bis(2-aminophenoxy)ethane, N,N′-di(2-benzyloxyethyl acetate),N,N′-acetic acid; 1,2-bis(2-aminophenoxy)ethane,N,N′-di(2-dodecyloxyethyl acetate), N,N′-diacetic acid;1,2-bis(2-aminophenoxy)ethane, N,N′-di[2-(2-dodecyloxyethoxy)-ethylacetate], N,N′-diacetic acid; and 1,2-bis(2-aminophenoxy)ethane, N,N′-di{2-[2-(2-dodecyloxyethoxy) ethoxy]-ethyl acetate}, N,N′-diacetic acid.